Despite extensive exposure to M. tuberculosis over long periods, some persons remain tuberculin-negative, suggesting that innate immunity controls the infection before T cells recognize M. tuberculosis antigens. We have found that NK cells from healthy donors lyse M. tuberculosis-infected mononuclear phagocytes through binding of NKp46 and NKG2D on NK cells to vimentin and ULBP1, respectively, on infected cells. Our preliminary data indicate that, in tuberculin-negative persons with heavy exposure to tuberculosis (heavily exposed PPD-s), NK cells lyse more infected monocytes than NK cells from other healthy donors. Monocytes from heavily exposed PPD-s also produce high concentrations of IL-15. Based on these data, we hypothesize that: (1) NKp46, NKG2D and their ligands mediate the enhanced lytic capacity of NK cells from heavily exposed PPD-s; (2) NKp46, NKG2D and their ligands are upregulated by IL-15-mediated signaling. To address these hypotheses, we propose the following aims. [unreadable] [unreadable] Aim 1. Determine if NKp46, NKG2D and their ligands contribute to the increased capacity of NK cells from heavily exposed PPD-s to lyse M. tuberculosis-infected cells. 1.1. Surface expression and neutralization of NK cell receptors and ligands. We will measure expression of NKp46, NKG2D vimentin and ULBP1 by flow cytometry. We will use siRNA and neutralizing antibodies to these receptors and ligands to determine their contribution to lysis of infected cells. 1.2. Mechanisms for upregulation of NK cell receptors and ligands. We will determine if upregulated NK cell receptors and ligands in heavily exposed PPD-s show increased mRNA expression or increased translocation of protein to the cell surface. 1.3. Do heavily exposed PPD-s show greater NK cell lysis of cells infected with other pathogens? We will evaluate the capacity of NK cells from heavily exposed PPD-s to lyse cells infected with Listeria, Cryptosporidium and Epstein Barr virus. We will also measure expression of NK cell receptors and ligands found to be upregulated in aim 1.1. [unreadable] [unreadable] Aim 2. Determine the contribution of IL-15 to the capacity of NK cells from heavily exposed PPD-s to lyse infected cells. 2.1. Effects of IL-15 on expression of NKp46, NKG2D, vimentin and ULBP1. We will use antibodies to IL-15 and its receptor, as well as siRNA, to determine if IL-15 increases expression of NK cell receptors and ligands in heavily exposed PPD-s, and to determine if it does so through soluble cytokine or by binding to IL-15R and signaling in trans to NK cells. 2.2. To delineate the signaling pathways by which IL- 15 increases expression of NK cell receptors and ligands. We will evaluate expression of STAT3, STAT5 and STAT6 in NK cells and infected monocytes by Western blot. Next, we will use siRNA and lentiviral vectors to alter STAT levels, and identify the IL-15-induced STATs that increase expression of NK cell receptors and ligands. These studies will provide insight into innate mechanisms of resistance to tuberculosis, which will be critical for development of vaccines that maximize innate immune responses. [unreadable] [unreadable] Tuberculosis is an infectious disease that kills 1.9 million people worldwide annually, and development of an effective vaccine is an urgent public health priority. Some persons have innate resistance to tuberculosis and do not become infected despite extensive exposure to infectious tuberculosis patients. This proposal will provide new insight into the immunologic mechanisms for this increased resistance, and harnessing of these mechanisms will be critical to develop a vaccine that maximizes innate resistance to tuberculosis. [unreadable] [unreadable] [unreadable] [unreadable]